Materials
HD5 and HD5-myr in this study were chemically synthesized via solid-phase peptide synthesis by CHINESE PEPTIDE (Hangzhou, China). The obtained peptides were purified by HPLC, and molecular weights were verified by MALDI-TOF MS (BRUKER ultrafleXtreme, Germany). PVA (26–99), with degree of alcoholysis 98–100%, was purchased from Anhui Wanwei Group Co., Ltd (Chao Hu, China). The Luria-Bertani (LB) medium was composed of 5 g yeast extract (Thermo Scientific Oxoid, USA), 10 g tryptone (Thermo Scientific Oxoid, USA) and 10 g NaCl (Sinopharm Chemical Reagent Co., Ltd.; China) per 1000 mL double distilled water (ddH2O). To generate solid LB agar plates, and additional 15 g agar powder (Sinopharm Chemical Reagent Co., Ltd.; China) was added to the above formula. All bacterial culture media were sterilized by autoclaving to remove possible biological contamination before use. The bacterial strains used in this project, including E. coli (25922), K. pneumoniae (13883), S. aureus (25923), A. baumannii (17978), P. aeruginosa (27853), MRSA (43300), were obtained from American Type Culture Collection (ATCC). The clinical isolates, carbapenems-resistant P. aeruginosa, were obtained from the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. The bacterial strains including imipenem/cilastatin sodium-resistant A. baumannii and vancomycin-resistant S. aureus used in this study were induced from A. baumannii (17978) and S. aureus (25923).
Animals
BALB/c and C57BL/6 mice used in this study were purchased from the Shanghai SLAC Laboratory Animal Center. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang University.
Synthesis of PVA@HD5-myr nonwoven fabrics
PVA was repeatedly cleaned with ultrapure water and soaked for 2 h at room temperature. Then PVA aqueous solution (16 %, wt/wt) was prepared by dissolving PVA granule in ultrapure water with vigorous stirring at 90 °C–100 °C and the pH of the solution was adjusted to 5.5–6.5 with aqueous solution of citric acid. After the solution was cooled down to 25 °C ± 2 °C, HD5-myr (14 ppm) was added to form the mixtures. The mixtures were stirred thoroughly to obtain homogeneous spinning dope. Before ejecting the fiber from the spinneret, the crosslinking agent Tyzor®LA (60 ppm) was added continuously through the metering pump. Then the spinning dope containing Tyzor®LA was made into PVA@HD5-myr fibers, which were dried and stretched in the spinning shaft containing hot air (45 °C–50°C). Next, the PVA@HD5-myr fibers were prepared into spunlaced nonwoven fabrics with a basis weight of 68 g m-2. Finally, PVA@HD5-myr nonwoven fabrics were sterilized by 60Co (15 kGy) irradiation.
SEM and EDS analysis of PVA and PVA@HD5-myr nonwoven fabrics
The physical morphologies and element composition of PVA and PVA@HD5-myr nonwoven fabrics were analyzed via the combined SEM and SEM-EDS. Firstly, PVA@HD5-myr was washed thoroughly with distilled water to remove the HD5-myr that was not covalently bound on the surface. Dried samples were sputter-coated with platinum using a sputter coater (Hitachi, Japan). The morphology of samples was examined using an SEM (ZEISS GeminiSEM 300, Germany), while elemental analysis (C, O, N for PVA and PVA@HD5-myr) was performed using BRUKER EDS (Germany) at 12 keV.
XPS analysis
The surface elemental composition of PVA and PVA@HD5-myr nonwoven fabrics were analyzed using an X-ray photoelectron spectrometer (Thermo Scientific ESCALAB 250Xi, USA). Monochromatic Al Kα X-ray (hv = 1486.6 eV) was employed for analysis. The analysis diameter of the samples was approximately 500 µm. The analysis depth of the samples was approximately 50–100 nm. The elemental atomic percentages of PVA (C, O), HD5-myr (C, O, N, S) and PVA@HD5-myr (C, O, N, S, Ti) were determined.
In vitro antimicrobial activity assay of PVA@HD5-myr nonwoven fabrics
The antimicrobial activity assay of PVA and PVA@HD5-myr nonwoven fabrics referred to the National Standards of P.R.C (GB 15979-2002) and was modified appropriately. Briefly, single colonies from LB agar plates were cultured in 5 mL LB medium. After growing for 6–8 h at 37 °C, 200 rpm in an incubator, the bacteria were washed thrice with PBS. To determine the bactericidal activity, 5 mL bacterial suspension was added into a conical flask containing 50 mL PBS, and the bacterial concentration was adjusted to 1 × 106 CFU mL-1. Three pieces of PVA or PVA@HD5-myr nonwoven fabrics (6 × 6 cm) were added into the conical flask and then incubated at 37 °C, 200 rpm for 1 h. Following incubation, the bacteria in the conical flask were counted. In some experimental groups, PVA@HD5-myr nonwoven fabrics were treated at 37 °C, 60% RH for 33 days before antimicrobial activity assay to test their stability. The experiments were repeated three times. The lower limit of detection was 100 CFU. To visualize the data on a logarithmic scale, a value of 100 CFU was assigned when no bacterial growth occurred.
Bacterial morphology under SEM
The morphologies of carbapenems-resistant P. aeruginosa, imipenem/cilastatin sodium-resistant A. baumannii and MRSA after treatment with PVA or PVA@HD5-myr nonwoven fabrics were observed under a Nova Nano 450 field-emission electron microscope (Thermo FEI, USA). Bacteria (OD600 = 1.0) were added to PVA or PVA@HD5-myr nonwoven fabrics and incubated at room temperature for 1 h. A 2.5% glutaraldehyde solution was added for fixation overnight at 4 °C. Then, the samples were washed with PBS three times and then incubated with 1% OSO4 solution for 1.5 h. After washing another three times with PBS, dehydration was performed using a gradient concentration of ethanol solution (one time in 30, 50, 70, 80, 90, and 95% for 15 min and two times in 100% for 20 min each). After drying in a LEICA EM CPD300 critical point dryer (LEICA, Germany), the samples were coated with platinum before SEM analysis.
LIVE/DEAD assay to examine bacterial viability
Bacteria suspension (final concentration of 5 × 107 CFU mL-1) was added into a conical flask containing 50 mL PBS. PVA or PVA@HD5-myr nonwoven fabrics were added into the conical flask and then incubated at 37 °C, 200 rpm. After 1 h, the bacteria were pelleted and resuspended with 0.9% NaCl. Then bacteria were stained with LIVE/DEAD BacLight Bacterial Viability Kits (including PI and SYTO 9 dye; Invitrogen L13152, USA) for 15 min and observed with a laser scanning confocal microscopy (Olympus FV3000, Japan)58. The excitation/emission maxima for these dyes are about 480/500 nm for SYTO 9 stain and 490/635 nm for PI.
In vitro inhibition of biofilm formation by HD5 and HD5-myr
As described previously59, single colonies of bacteria (carbapenems-resistant P. aeruginosa, imipenem/cilastatin sodium-resistant A. baumannii, or MRSA) from LB agar plates were selected and cultured to mid-logarithmic growth phase in 5 mL of LB medium at 37 °C, 200 rpm. In the 96-well polypropylene plates (Corning, USA), bacteria (a final OD600 of 0.01) were exposed to HD5 and HD5-myr in Tryptic Soy Broth (TSB) medium (Solarbio, China) supplemented with 1% (wt/vol) glucose (Sinopharm Chemical Reagent Co., Ltd.; China). The final concentrations of HD5 and HD5-myr ranged from 3.125 to 50 μg mL-1. As an untreated control, bacteria were exposed to a TSB medium supplemented with 1% (wt/vol) glucose without HD5 or HD5-myr. After 48 h incubation at 37 °C, spent growth medium and non-adhered planktonic cells were removed by three washes with PBS. Biofilms were stained with 0.1% (wt/vol) crystal violet (Aladdin, China) for 30 min at room temperature with gentle shaking on an orbital shaker, washed again, and solubilized with 75% ethanol for 30 min. The absorbance at 595 nm was determined on a microplate spectrophotometer to measure biofilm mass.
In vitro antibiofilm effect of PVA@HD5-myr nonwoven fabrics
The antibiofilm effect of PVA@HD5-myr nonwoven fabrics was evaluated against bacteria of imipenem/cilastatin sodium-resistant A. baumannii, carbapenems-resistant P. aeruginosa and MRSA. Briefly, microbial suspension (1 mL, 3 × 106 CFU mL-1) was cultured with PVA or PVA@HD5-myr (1 × 1 cm) in a 24-well plate (Corning, USA) and incubated for 48 h at 37 °C. After that, all samples were taken out and washed thoroughly with PBS to remove the plankton bacteria. Then PVA and PVA@HD5-myr were added into 2.5% glutaraldehyde solution for fixation overnight 4 °C. Samples were prepared using the above method and observed with SEM.
Murine wound bacterial infection model
Seven to eight weeks of male BALB/c mice, weighing 22–24 g, were used in this experiment. The mice were individually raised in cages at a standardized environment and night cycle and given an autoclaved rodent diet and sterile water and then randomly divided into three groups, including the sham-operated group, the PVA treatment group, and the PVA@HD5-myr treatment group. Mice were weighed and anesthetized with an intraperitoneal injection of ketamine/xylazine (100 and 10 mg kg-1). Then, the hair was shaved from the backside of the mice using a depilatory cream. The mice of the sham-operated group did not undergo cutaneous wound surgery, while the other groups underwent cutaneous wound surgery after anesthesia. A full-thickness back skin wound (1 × 1 cm) of mice was cut using a surgical scalpel. Two pieces of PVA or PVA@HD5-myr nonwoven fabrics (2 × 2 cm) covered the wound bed and were fixed with thin transparent dressings (SIMP, Shanghai, China). To verify the effect of nonwoven fabrics against bacterial infection, 1 × 106 CFU of MRSA was added. After 15 days, the mice were weighed again. We photographed the wound areas to analyze wound healing and all mice in the three groups. Skin tissue samples were excised and used for bacterial burden measurement, H&E staining and Masson’s trichrome staining. Skin histopathological scores were based on the five aspects described previously60, including epidermal regeneration (0, none; 1, mild; 2, moderate; 3, complete), neovascularization (0, absence; 1, occasional presence; 2, light scattering; 3, abundance), collagen deposition (0, 0–10% positive area; 1, 10–20% positive area; 2, 20%-30% positive area; 3, 30%-40% positive area; 4, å 40% positive area), granulation tissue thickness (0, none; 1, mild; 2, moderate; 3, complete) and inflammatory infiltration (0, severe; 1, moderate; 2, mild; 3, almost absent). The positive area of skin collagen deposition was analyzed using ImageJ software. On the third day after the injury, bacterial biofilm formation on the PVA or PVA@HD5-myr dressings and on the skin surface of mice were examined under SEM.
Bacterial burden measurement
Skin tissue samples collected 15 days post-wounding were homogenized using a homogenizer (TissuePrep, Tianjin, China) and diluted in sterile PBS. The homogenate was diluted and then inoculated on LB plates. The number of bacteria was counted after overnight incubation at 37 °C, and the results were expressed as CFU per gram of tissue. To visualize the data on a logarithmic scale, a value of 1 CFU was assigned when no growth occurred61.
SPR
SPR binding experiments were carried out on a BIAcore 3000 system. The assay buffer adopted in this assay was HBS-EP, containing 0.15 M NaCl, 3 mM EDTA, 0.05% (vol/vol) surfactant P20, and 0.01 M HEPES (pH 7.4). HD5 and HD5-myr dissolved in 10 mM NaAc (pH 4.5) were immobilized on a CM5 sensor chip using the amine-coupling chemistry recommended by the manufacturer’s instructions. A series of concentrations of LPS (Escherichia coli O111:B4; Sigma-Aldrich L2630, USA) was introduced into the flow channels at 30 mL min-1 in the assay buffer. The molecular mass for E. coli O111:B4 LPS was considered to be 10 KDa62. Association and dissociation were assessed for 3 min. Resonance signals were corrected for non-specific binding by subtracting the background of the control flow channel. After each analysis, the CM5 sensor chip surfaces were regenerated with 10 mM citric acid/1 M NaCl (pH 2.95) and 10 mM Gly/HCl solution (pH 2.5) for 400 s at a flow rate of 30 μL min-1, and equilibrated with the buffer before next injection. The manufacturer-supplied software for BIAcore 3000 was applied to analyze the signal curves and calculate the association constant (Ka), dissociation constant (Kd), and affinity constant (KD). Data was analyzed using GraphPad 7.0 software.
Mouse wound acute inflammation model
Seven to eight weeks of male BALB/c mice were randomly divided into four groups, the sham-operated group, the control group, the PVA treatment group, and the PVA@HD5-myr treatment group. Each group of mice except the sham-operated group were treated with the same trauma described above. The PVA and PVA@HD5-myr treatment group were covered with two pieces of PVA and PVA@HD5-myr nonwoven fabrics (2 × 2 cm), respectively, and then thin transparent dressings (SIMP, Shanghai, China) were fixed. To verify the effect of nonwoven fabrics against bacterial endotoxin, LPS (20 μg in 50 μL PBS) was added to the wound site. After 6 h, the wound areas were washed six times with 50 μL of endotoxin-free water (Sigma-Aldrich, USA) each time. Endotoxin was removed from all instruments used in this experiment.
Survival analysis of mice
A full-thickness back skin wound (1 × 1 cm) of BALB/c mice was constructed. Then two pieces of PVA or PVA@HD5-myr dressings (2 × 2 cm) covered the wound bed and were fixed with thin transparent dressings. The skin wound of mice in control group was not covered with dressing. Then P. aeruginosa (ATCC 27853, 1 × 108 CFU in 50 µL PBS) was added. The survival of mice was continuously monitored for 72 h.
Limulus amebocyte lysate (LAL) assay
According to the manufacturer’s instructions, the endotoxin content in skin washing fluids was measured by a PierceTM Chromogenic Endotoxin Quant kit (Thermo Scientific, USA).
Cytokine Measurement
The skin washing fluids were used to analyze TNF-α, IL-6 and IL-1β protein levels using enzyme-linked immunosorbent assay (Novus Biologicals, Bio-Techne China) according to the manufacturer’s recommendations.
